Anti-RBCK1 antibody [EPR28157-53] - BSA and Azide free

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-RBCK1 antibody [EPR28157-53] - BSA and Azide free (AB309105)

This data was developed using ab309104, the same antibody clone in a different buffer formulation. Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HepG2 (human hepatocellular carcinoma epithelial cell) cells labelling RBCK1 with ab309104 at 1/50 (10.14 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green). Confocal image showing mostly cytoplasmic staining in HepG2 cell line. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-RBCK1 antibody [EPR28157-53] - BSA and Azide free (AB309105)

This data was developed using ab309104, the same antibody clone in a different buffer formulation. Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HepG2 (human hepatocellular carcinoma epithelial cell) cells labelling RBCK1 with ab309104 at 1/500 dilution (0.1 ug)/Red (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-RBCK1 antibody [EPR28157-53] - BSA and Azide free (AB309105)

This data was developed using ab309104, the same antibody clone in a different buffer formulation. Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling RBCK1 with ab309104 at 1/500 dilution (0.1 ug)/Red (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• WB

Supplier Data

Western blot - Anti-RBCK1 antibody [EPR28157-53] - BSA and Azide free (AB309105)

This data was developed using ab309104, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : 5% NFDM/TBST. The expression molecular weight observed is consistent with what has been described in the literature (PMID : 31209050, PMID : 27006117). The higher MW(57kd) band is the ubiquitylated form of RBCK1(PMID : 31209050). Bands below 57kd are cleaved RBCK1(PMID : 27006117).

All lanes:

Western blot - Anti-RBCK1 antibody [EPR28157-53] (<a href='/en-us/products/primary-antibodies/rbck1-antibody-epr28157-53-ab309104'>ab309104</a>) at 1/1000 dilution

Lane 1:

A549 (human lu carcinoma epithelial cell)whole cell lysate at 20 µg

Lane 2:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 3:

C2C12 (mouse myoblast) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 57 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-RBCK1 antibody [EPR28157-53] - BSA and Azide free (AB309105)

This data was developed using ab309104, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : 5% NFDM/TBST The expression molecular weight observed is consistent with what has been described in the literature (PMID : 31209050, PMID : 27006117). The higher MW(57kd) band is the ubiquitylated form of RBCK1(PMID : 31209050). Bands below 57kd are cleaved RBCK1(PMID : 27006117). The blot of lanes1, 2 and 6-8 were developed using a high sensitivity ECL substrate. Exposure time : Lanes1, 2 and 6-8 : 180 seconds; Lane3-5 : 48 seconds

All lanes:

Western blot - Anti-RBCK1 antibody [EPR28157-53] (<a href='/en-us/products/primary-antibodies/rbck1-antibody-epr28157-53-ab309104'>ab309104</a>) at 1/1000 dilution

Lane 1:

Human hypothalamus tissue lysate at 20 µg

Lane 2:

Human spinal cord tissue lysate at 20 µg

Lane 3:

Mouse brain tissue lysate at 20 µg

Lane 4:

Mouse spleen tissue lysate at 20 µg

Lane 5:

Mouse kidney tissue lysate at 20 µg

Lane 6:

Rat brain tissue lysate at 20 µg

Lane 7:

Rat spleen tissue lysate at 20 µg

Lane 8:

Rat kidney tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 57 kDa

false

• WB

Supplier Data

Western blot - Anti-RBCK1 antibody [EPR28157-53] - BSA and Azide free (AB309105)

This data was developed using ab309104, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : 5% NFDM/TBST. The expression molecular weight observed is consistent with what has been described in the literature (PMID : 31209050, PMID : 27006117). The higher MW(57kd) band is the ubiquitylated form of RBCK1(PMID : 31209050). Bands below 57kd are cleaved RBCK1(PMID : 27006117). In Western blot, anti- H3 antibody (ab176842) loading control staining at 1/100000 dilution.

All lanes:

Western blot - Anti-RBCK1 antibody [EPR28157-53] (<a href='/en-us/products/primary-antibodies/rbck1-antibody-epr28157-53-ab309104'>ab309104</a>) at 1/1000 dilution

Lane 1:

HepG2 (human hepatocellar carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HepG2 transfected with siRNA specifically targeti RBCK1 whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 57 kDa

false

Exposure time: 59s

• WB

Lab

Western blot - Anti-RBCK1 antibody [EPR28157-53] - BSA and Azide free (AB309105)

This data was developed using the same antibody clone in a different buffer formulation (ab309104).

Western blot : Anti-RBCK1 antibody [EPR28157-53] (ab309104) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab309104 was shown to bind specifically to RBCK1. A band was observed at 58 kDa in wild-type A549 cell lysates with no signal observed at this size in RBCK1 knockout cell line. To generate this image, wild-type and RBCK1 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-RBCK1 antibody [EPR28157-53] (<a href='/en-us/products/primary-antibodies/rbck1-antibody-epr28157-53-ab309104'>ab309104</a>) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

RBCK1 knockout A549 cell lysate at 20 µg

Lane 3:

HepG2 cell lysate at 20 µg

Lane 4:

MCF7 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 58 kDa

false